Thiamin is an essential component of the human diet and is added to many commercial foods (RDA = 1.4 mg). Deficiency in this vitamin results in a neurological disease called beri-beri. At the molecular level, thiamin dependent enzymes play a particularly important role in carbohydrate metabolism and include transketolases, alpha-ketoacid decarboxylases, alpha-ketoacid oxidases, acetolactate synthase and glyoxalate synthase. The long range goals of our research are to understand the mechanistic enzymology and regulation of the thiamin biosynthetic pathway in Escherichia coli. These studies are significant for three reasons. Firstly, since this vitamin is a required component of the human diet, it is important to understand how it is biosynthesized. Secondly, from the perspective of basic science, the biosynthetic pathway involves an unusually large amount of novel mechanistic chemistry. Lastly, our studies will facilitate the construction of overexpression strains that may be of use for the commercial production of thiamin or its components (2,000 tons/year produced by total synthesis). In this grant, we will carry out a complete mechanistic characterization of the enzyme involved in the coupling of the thiazole and the pyrimidine moieties of thiamin (thiamin phosphate pyrophosphorylase). In parallel, we will carry out a series of experiments designed to identify the reactions involved in the thiazole biosynthesis. Once we have identified specific reactions, we will initiate systematic mechanistic studies on each enzyme.